Electrophoretic separations as a means of purifying proteins and separation complex protein mixtures have assumed many different forms. The separations vary in the composition of separation medium, the geometrical configuration of the medium, the manner in which mobility through the medium is achieved, and the parameter on which separation is based. One type of electrophoretic separation which is particularly useful for protein separations is a separation performed in a linear separation medium whose pH varies with the distance along the medium. A prominent example of a separation process that utilizes this type of medium is isoelectric focusing, a process by which proteins or other amphoteric substances migrate under the influence of an electric field along the pH gradient, each species continuing its migration until it reaches a location at which the pH in the medium and the isoelectric point of the species are equal. When this condition is achieved, the net charge on the species and hence the driving force for migration are zero, and migration ceases. By the completion of the procedure, the various species in a sample occupy positions in discrete, non-moving ("isoelectrically focused") zones along the pH gradient that correspond to their isoelectric points.
Isoelectric focusing may constitute the entire separation process, in which case the components of the sample mixture are identified by the location of the zones (in comparison to a standard) and the amount of each component is determined by the relative intensity of its zone as detected by standard detection methods. Isoelectric focusing can also serve as the first dimension of a two-dimensional separation, the second dimension being performed by placing the linear medium with its isoelectrically focused zones along one edge of a two-dimensional (slab-shaped) separation medium, preferably one that does not contain a pH gradient or one in which separation is performed by way of a separation parameter other than the isoelectric point of the species. An electric field is then imposed in a direction transverse to the linear medium, causing migration of the contents of each focused zone out of that medium and into the slab-shaped medium along parallel paths, the contents of each zone thereby undergoing further separation.
The most convenient means of achieving and maintaining the pH gradient needed for isoelectric focusing is the use of a dimensionally stable medium consisting of a molecular matrix to which functional groups have been attached that are either charged or chargeable by the placement of the medium in an electric field. Strips of solid material that contain such groups are commonly referred to as "immobilized pH gradient" ("IPG") strips. Examples of such strips and their composition and structure are described by Rosengren et al. in U.S. Pat. No. 4,130,470, issued Dec. 19, 1978. The solid material that forms the matrix of the strip is either a granular, fibrous, or membrane material, or a gel. Examples of suitable materials are polyacrylamide, cellulose, agarose, dextran, polyvinylalcohol, starch, silica gel, and polymers of styrene divinyl benzene, as well as combinations of these materials. Examples of positively charged or chargeable groups are amino groups and other nitrogen-containing groups. Examples of negatively charged or chargeable groups are carboxylic acid groups, sulfonic acid groups, boronic acid groups, phosphonic or phosphoric acid groups, and esters of these acids. The groups are immobilized on the matrix by covalent bonding or by any other means that will secure the positions of the groups and prevent their migration when exposed to an electric field or to the movement of fluids or solutes through the strip. When the matrix is a polymer, for example, a typical means of immobilization, is the inclusion of charged monomers to copolymerize with the uncharged monomers that form the bulk of the polymer or the inclusion of charged crosslinking agents. Copolymerization or crosslinking can be performed in a manner that will result in a monotonic increase or decrease in the concentration of the charged or chargeable groups, thereby producing the gradient. Although IPG strips are formed in hydrated condition, they are typically dehydrated once formed and are supplied to users in this condition. Rehydration for use is conveniently achieved by the sample itself, which is applied to the strip and the strip permitted to stand for a sufficient period of time to achieve full rehydration.
While IPG strips offer the advantage of a stable and well-controlled pH gradient and require only rehydration to be ready for use, their use poses certain difficulties. Once a strip is rehydrated, for example, care must be taken to assure that the strip does not suffer dehydration during use by losing water to the atmosphere. Since the strip is generally not contained in a capillary or length of tubing or other enclosure that would shield it from atmospheric exposure, dehydration is typically prevented by covering the strip with an electrically insulating, water-immiscible liquid such as mineral oil, and keeping the strip covered during isoelectric focusing. Furthermore, contact of the two ends of the strip with electrodes must be made and maintained through the mineral oil. In addition, once isoelectric focusing has been performed, the mineral oil must be completely removed from the strip before the strip can be used in a second dimension separation, since residual mineral oil will interfere with the electrical continuity between the strip and the slab gel.